1-ANILINO-8-NAPHTHALENE SULFONATE BINDING SITE ON HUMAN ERYTHROCYTE MEMBRANE USING FLUORESCENCE LIFETIME AND POLARIZATION

Abstract

It was shown that the human erythrocyte ghost membrane had two kinds of binding site for 1-anilino-8-naphthalene sulfonate (ANS) from binding kinetics. In measuring the fluorescence lifetime of ANS in the human erythrocyte ghost membrane suspension, two kinds of fluorescence lifetime were obtained : τ₁=15 ns at low ANS concentration and τ₂=8.4 ns at high ANS concentration. Comparing the results obtained from binding kinetics with those from fluorescence lifetime, it was considered that more hydrophobic binding site with fluorescence lifetime τ₁ contained the binding site with high and low binding constant and less hydrophobic binding site with fluorescence lifetime T₂ corresponded to the binding site with low binding constant. From the results of binding kinetics of ANS to the erythrocyte membrane and the extracted membrane components (proteins and lipids), and those of the rotational relaxation time obtained from the ANS polarization in the erythrocyte membrane suspension, it was shown that the binding sites of ANS in the human erythrocyte ghost membrane were mainly composed of proteins at low ANS concentrations and lipids at high ANS concentrations.